Histamine measuring apparatus and a histamine measuring method

ABSTRACT

Histamine may be quantitatively measured by performing the following steps. First, an oocyte that expresses histamine receptors is held in a recess formed at the bottom of a vessel. Then, first and second electrodes are inserted into the oocyte. Subsequently, the membrane potential of the oocyte is measured by using the first electrode to stabilize this membrane potential at a predetermined level by driving a current through the second electrode using circuitry for clamping the membrane potential of the oocyte. A sample is then infused into a fine reacting tube having an antigen immobilized on its inner surface together with some buffer solution to promote a histamine releasing reaction. The solution containing histamines that is released in the fine reacting tube is transferred to the vessel to make contact with the oocyte in the vessel. Finally, an electric response of the oocyte caused by the contact with the solution is detected and the concentration of the histamine released in the histamine releasing reaction in the fine reacting tube is determined. The entire blood or mast cell suspension may be used as a sample without pretreatment.

This is a divisional application of U.S. Ser. No. 09/604,512, filed Jun.27, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to a histamine measuring apparatus anda histamine measuring method for quantitatively measuring histaminepresent in blood or mast cells suspensions.

2. Description of the Prior Art

A histamine releasing test is a quantitative analysis of to histamineextracellularly released by stimulating lewcocytes (white blood cells)in the blood or mast cells in the mucosa and promoting releasinghistamine contained therein outside the cells. The histamine releasetest has been reported to be a method useful in identifying someallergens in some allergic disorders and diseases (c.f., Prior Art 1:Tabe, kazuaki: histamine release test—diagnosis of allergies, SRL HOKAN,vol 21, pp. 17-22 (1997).

The known measurements of freed histamine (histamine released in freestate) includes, for example, a method using fluorescent HPLC(fluorescent high performance liquid chromatography) to purify thehistamine to react it with a fluorescent reagent in order to measure theamount of fluorescence emitted from the fluorescent reagent reacted withthe histamine (c.f. , Prior Art 2: Japanese Patent Laid-Open No.H6-331619), a method using glass fibers to purify the freed histamine toreact it with a fluorescent reagent in order to measure the amount offluorescence emitted from the fluorescent reagent coupled with the freehistamine (c.f., Prior Art 3: Japanese Patent Laid-Open No. H10-170514,Japanese Patent Laid-Open No. 10-62415), competitive immunoassayscommercially available from ICN Pharmaceuticals or Immunotech, and anELISA (c.f., Prior Art 4).

SUMMARY OF THE INVENTION

In general, the measurement of histamine may require:

(1) direct analysis of histamine without using labeling thereof;

(2) direct quantitative measurement of histamine without pretreatment;

(3) quick delivery of results; and

(4) specificity to the histamine.

Prior Art methodologies as have been described above are all indirect,quantitative measurement methods of histamine, which isolate thehistamine from a sample, and label the isolated histamine with afluorescent reagent to measure the labeled histamine reacted with afluorescent reagent. All of the Prior Art methodologies requires complexpretreatment such as purification of samples, isolation of histamine,and labeling of histamine with a fluorescent reagent, and also requiresfor hours to obtain a quantitative results of histamine analysis.

The primary object of the present invention is in general to provide ahistamine measuring apparatus and a histamine measuring method, whichmay satisfy the requirements listed above of histamine measurement.

In accordance with the histamine quantitative analyzing apparatus andmethod of the present invention, histamine quantitative analysis will beachieved by:

providing cells with histamine receptor being expressed;

adding a sample having been stimulated by an antigen to the cellsexpressing the receptor; and

detecting the resulting electrical response of cells.

For example, a predetermined amount of antigen will be added to a sampleof collected whole blood, the sample with antigen will be incubated for10 to 30 minutes at 37° C. to promote an allergic reaction (antigenchallenge) therebetween to release the histamine into the plasma. It maybe preferable to gently shake the blood sample while promoting theallergic reaction. The period of time of reaction described here isindicated by way of example. The reaction time may vary depending on theoptimized allergic reaction.

Examples of sample include, whole blood specimen, leucocytes in theblood, and mast cell suspensions in which the mast cells of mucosaltissue are cultured.

An allergic reaction may be invoked in general by the binding of anallergen with a corresponding IgE present in the cell membrane of cellscontributing to the allergic reaction such as mast cells, basophils, inthe sample blood. The binding may trigger a reaction of releasing arelevant chemical mediator (chemical transmitter) such as histamine inthe cells contributing to the allergy reaction. In the descriptionhereinbelow, the reaction of releasing histamine will be referred to as“histamine release (releasing) reaction.”

The concentration (A) of histamine extracellularly released by thehistamine releasing reaction may be determined by using a calibrationcurve thereof. The calibration curve may be obtained by stimulatingcells with the histamine receptor preliminary expressed by means of aplurality of known concentrations of histamine to detect the cellularelectric responses at respective concentration of histamine and may beexpressed as a relation between a plurality of known concentrations ofhistamine and detected electric responses.

The ratio of histamine release α (%) may be determined by

α=100×(A−V)/B

where B is a histamine concentration given by the quantitativemeasurement of histamine released from the sample after freezing andthawing the sample having stimulated by the antigen, C is aconcentration of free histamine released in non stimulated state byadding some buffer solution instead of the antigen to the sample.

The concentration of released histamine (C) designates to a releasedhistamine concentration by the stimulation with the buffer, which isso-called “concentration for the correction of background level”.

The apparatus and method in accordance with the present invention allowsthe histamine released in the sample to be directly and quantitativelyanalyzed by using cells with histamine receptors expressed. The cellsexpressing histamine receptors may identify specifically and directlythe histamine, even in case in which derivatives of histamine orhistamine-like compounds are present in the sample, or in case in whichthere is a trace of histamine contained in the sample. The apparatus andmethod in accordance with the present invention, accordingly, may allowdirect quantitative analysis of histamine to be performed in a shortertime, with no complex pretreatment of samples which may require longtime.

An exemplary configuration of the present invention will be summarizedbelow. The oocyte expressing the histamine receptors is held in therecess formed at the bottom of a vessel. A first electrode and a secondelectrode will be inserted into the oocyte to determine the membranepotential of oocyte by the first electrode, and then maintain thepotential of oocyte membrane to a predetermined level by flowingelectric current through the second electrode, by means of a circuitryfor maintaining the potential of oocyte membrane. A sample will be flewthrough together with some buffer into a fine reacting tube with anantigen fixed on the inner surface of tube wall to promote the histaminereleasing reaction. Thereafter the solution containing free histaminewill be flew through a flowing tube into the vessel to make contact withthe oocyte in the vessel. The electric response of the oocyte caused bythe contact with the solution will be detected by the potentialmaintainer (clamping) circuit in order to determine the concentration offree histamine released by the histamine releasing reaction. The wholeblood, or suspension may be used as the sample, without the need for anypretreatment.

In accordance with the present invention, the concentration of histaminemay be determined in a shorter time, without the need for anypretreatment, so that the process steps for quantitative analysis, thenumber and amount of reagents may be reduced to minimum and the cost andthe time needed for the measurement may be significantly saved.

In the following description an oocyte of Xenopus lavis (African clawedfrog) will be used for the cell expressing the histamine receptors, byway of example. It should be understood that the present invention maybe applicable by using any other types of cell.

Also in the following description of preferred embodiments, an exemplarycase will be described in which the electric response of the cell withhistamine receptors expressed will be detected by identifying histaminewith the histamine receptors expressed on the cell membrane and bymeasuring the change in the membrane potential along with the open andclose of chloride ion channel caused by the intracellular signaltransduction. It is to be understood that the preferred embodimentsdescribed herein are presented and described for the purpose ofillustrating the principle of the present invention and are not intendedto be limiting. For example, it should be recognized that the presentinvention may be achieved by using the cellular response derived fromthe receptor stimulation of any other kinds such as those described inthe Japanese Patent Laid-Open No. H11-083785 (Prior Art 5), or by usingany other detecting methods.

Additional objects and advantages of the invention will be set forth inpart in the description which follows and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention may be realized and attained bymeans of the instrumentalities and combinations particularly pointed outin the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram illustrating an apparatus in accordancewith one preferred embodiment of the present invention, which is ahistamine measuring apparatus for quantitatively analyzing the histamineby using the oocyte with histamine receptors expressed;

FIG. 2 shows a schematic diagram illustrating an exemplary analysis ofhistamine concentration and electric response of the oocyte bothobtained by the apparatus shown in FIG. 1;

FIG. 3A shows a schematic diagram illustrating an exemplary analysis ofelectric response of oocyte, induced by the blood sample containinghistamine, and obtained by using the apparatus shown in FIG. 1;

FIG. 3B shows a schematic diagram illustrating an exemplary analysis ofelectric response of oocyte, induced by the blood sample containing nohistamine, and obtained by using the apparatus shown in FIG. 1;

FIG. 4 shows a protocol of histamine analysis method in accordance witha preferred embodiment of the present invention;

FIG. 5 shows a result of histamine analysis using a whole blood sample,obtained by using the apparatus shown in FIG. 1; and

FIG. 6 shows a schematic diagram of an apparatus for measuring histaminein accordance with another preferred embodiment of the presentinvention, illustrating the overview of apparatus for quantitativelyanalyzing histamine by using the oocyte with histamine receptorsexpressed and a whole blood sample.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following description of the preferred embodiments, the histaminemeasuring apparatus and the histamine measuring method in accordancewith the present invention will be described in greater details withreference to the quantitative analysis of histamine in the whole bloodsample by using the oocyte with histamine receptors expressed.

A whole blood sample stimulated by the antigen will be added to theoocyte with histamine receptor expressed to detect the resultingelectric response of the oocyte. A predetermined amount of antigen willbe added to the collected whole blood sample (some buffer may beintroduced if the whole blood sample is too viscous), and the wholeblood sample and the antigen will be incubated at a temperature rangingfrom 30° C. to 45° C. for 10 to 30 minutes for promoting the allergyreaction therebetween in order to release histamine into the plasma.

The concentration of histamine (A) released into the plasma from theblood cells in the sample caused by the histamine releasing reaction maybe determined by means of a calibration curve. The calibration curve maybe obtained by stimulating cells with the histamine receptor preliminaryexpressed by means of a plurality of known concentrations of histamineto detect the cellular electric responses at respective concentration ofhistamine and may be expressed as a relation between a plurality ofknown concentrations of histamine and detected electric responses.

The ratio of histamine release α (%) may be determined by

α=100×(A−C)/B

where B is a histamine concentration given by the quantitativemeasurement of histamine released from the cells in the sample bloodafter freezing and thawing the sample stimulated by the antigen, C is aconcentration of released histamine in non stimulated state by addingsome buffer instead of the antigen into the blood sample.

The concentration of released histamine in non-stimulation C designatesto the concentration of free histamine released by the stimulation ofbuffer, i.e., so-called “concentration of correction of background”.

The histamine analysis as have been described above may be carried outwithin approximately 20 to 50 minutes, including the calibrationmeasurement for determining the calibration curve to be used. In case inwhich the calibration curve has been determined in advance, then thehistamine measurement will be completed at most in 30 seconds for eachsample.

In this preferred embodiment of the apparatus and method in accordancewith the present invention, quantitative measurement of histaminereleased from the cells in the whole blood to the plasma may be directlycarried out. If the histamine derivatives or histamine-like compoundsare present in the cells or plasma of whole blood sample, or if theamount of histamine to be released into the plasma from the cells ofwhole blood is very low, the oocyte with histamine receptors expressedwill specifically and directly identify the histamine. Accordingly theapparatus and method for measuring histamine in accordance with thepresent invention may directly and quantitatively analyze the releasedhistamine from the whole blood cells to the plasma without the need forany pretreatment of whole blood sample.

Since the apparatus and method of the preferred embodiment in accordancewith the present invention may detect the histamine by using the oocytewith histamine receptors expressed, the electric response of the oocytecaused by the physiological reaction induced at the time when the oocyteidentifies the presence of histamine may be directly detected as anelectric signal. The physiological reaction of oocyte is known to beoccurred within milliseconds, thus the time required for detecting thehistamine may be within one second.

The apparatus and method for measuring histamine of the preferredembodiment in accordance with the present invention may perform aquantitative measurement in significantly shorter time than any otherPrior Art techniques requiring at least one hour for the analysis.

A detailed description of some preferred embodiments embodying thepresent invention will now be given referring to the accompanyingdrawings.

FIG. 1 shows a schematic diagram illustrating an apparatus in accordancewith one preferred embodiment of the present invention, which is ahistamine measuring apparatus for quantitatively analyzing the histamineby using the oocyte with histamine receptors expressed;

In this embodiment the oocyte of Xenopus lavis (African Clawed Frog)will be used. The unfertilized oocytes (eggs) of Xenopus lavis aresubjected to be injected mRNA of histamine receptor. These oocytes willbe incubated for two or three days in a culture medium so as to causehistamine receptors to be expressed. The histamine receptors will beactive within the cell membrane of oocyte. When histamine binds to thehistamine receptors, the information will be transferred inside theoocyte through the mediator (second messenger) already present in theoocyte.

As the result of a plurality of chemical mediation inside the oocyte,intracellular calcium ions are raised in the oocyte, opening thechloride ion channel of the oocyte cell membrane. This reaction willcause the change in potential of cell membrane inside and outside theoocyte.

In the present preferred embodiment of the present invention the changein the electric response of oocyte before and after the binding ofhistamine to the histamine receptors, namely, the change in potentialsbetween the inside and the outside of oocyte cell membrane, will bedetected as the change in electric current response.

In the present preferred embodiment of the present invention a method ofwhole cell clamp will be used in which a feedback circuitry will beused, which may flow current through the oocyte in the direction ofsuppressing the difference of membrane potential at the moment of changein potential of membrane of oocyte to maintain the potential to apredetermined constant holding potential.

As shown in FIG. 1, at the recess 16 (not shown) at the bottom of avessel 1 fulfilled with buffer solution 23 for the oocyte, an oocyte 2with the histamine receptors expressed will be held. The fine tips ofglass electrodes 3 and 4, filled with KCl of 3 mole. and containing anAg wire coated with AgCl will be inserted into the oocyte 2. After theinsertion, each of the tips of glass electrodes 3 and 4 will be fixedlyheld by is an apparatus for holding electrodes, which is not shown inFIG. 1, but is provided correspondingly for respective electrode.

The potential of the glass electrode 3 will be transmitted to thedifferential amplifier 6 and the recorder 7 and recorded therein as thedifference in potential to an external electrode 5. The glass electrode3 may be an electrode for measuring the potential in the membrane ofoocyte, while the glass electrode 4 may be used for maintaining themembrane potential of the oocyte to a predetermined constant level of−60 mV.

The differential amplifier 6 will apply the differential current betweenthe signal transferred from the glass electrode 3 to the differentialamplifier 6 and the holding potential in the whole cell clamping methodto the oocyte 2 through the glass electrode 4. in this manner, when thehistamine 8 will be dropped onto the oocyte 2 with the potential of cellmembrane held at a constant level of −60 mV, by means of for example amicro-syringe or a pipette 9, the electric response (current response10) of the oocyte 2 may be obtained. The current response 10 will bedescribed in greater details below.

FIG. 2 shows a schematic diagram illustrating an exemplary analysis ofhistamine concentration and electric response of the oocyte bothobtained by the apparatus shown in FIG. 1. The abscissa is the knownconcentration of histamine (nM) of the samples. The ordinate is theamount of change in potential between the inside and outside of theoocyte before and after the binding of histamine to the histaminereceptors, measured as the change of current (μA).

As shown in FIG. 2, it is clear that the current change is in positivecorrelation with the concentration of histamine in the range ofhistamine concentration from 20 nM to 200 nM. This correlation(calibration curve) may be used for measuring a sample containinghistamine at an unknown concentration by using the apparatus of thepresent invention shown in FIG. 1 so as to determine the unknownconcentration of histamine.

FIG. 3A and FIG. 3B show schematic diagrams illustrating an exemplaryanalysis of electric current response of the oocyte, induced by thewhole blood sample containing histamine (FIG. 3A) and by the bloodsample containing no histamine (FIG. 3B) and obtained by using theapparatus shown in FIG. 1.

As shown in FIG. 3A, when at the moment as shown by the vertical arrow,the whole blood sample containing histamine is added to the oocyteexpressing histamine receptors, the current response will be reached atthe maximum value within one second, then will decrease graduallytogether with the elapsed time to disappear approximately 30 secondsafter.

On the other hand, as shown in FIG. 3B, when the whole blood samplecontaining no histamine is added to the oocyte expressing histaminereceptors at the moment as shown by the vertical arrow, no change incurrent response will be observed. This indicates that the change incurrent response as shown in FIG. 3A is based on the reaction betweenthe histamine and histamine receptors, rather than the change ofresponse caused by the impurity of whole blood sample. When applying themaximum value of the current response shown in FIG. 3A to thecorrelation (calibration curve) shown in FIG. 2, it can be seen thatthis maximum value of the current response may be caused by thehistamine of concentration of approximately 100 nM. The outlinedvertical arrow shown in FIG. 3A indicates the change in current beforeand after adding a whole blood sample containing histamine to the oocyteexpressing histamine receptors.

As can be seen from the foregoing description, in accordance with thepresent invention, a whole blood sample may be used for quantitativeanalysis of histamine without needs for isolation, generator andlabeling. As have been described above, by measuring the change inmembrane potential as the change in current passing therethrough, alongwith the opening and closing of chloride ion channel caused byintracellular chemical mediator after identifying histamine by an oocyteexpressing histamine receptors, the measurements for quantitativeanalysis of histamine may be obtained within one second after the oocyteidentifies histamine.

EMBODIMENTS

[first embodiment]

By stimulating oocytes expressing histamine receptors with a pluralityof known concentrations of histamine to detect the change in current ashave been described above, the correlation (calibration curve) betweenthe change in current and the concentrations of histamine as shown inFIG. 2 may be predetermined.

FIG. 4 shows a protocol of histamine analysis method in accordance witha preferred embodiment of the present invention. The protocol will bedescribed below in greater details with reference to FIG. 4:

(1) A whole blood sample 11 will be collected from vein of a humanelbow, using heparin as anticoagulant.

(2) A predetermined amount of antigen 12 such as cedar pollen will beadded to the collected whole blood sample 11.

(3) The whole blood sample 11 will be incubated for approximately 30minutes at 37° C. to promote histamine releasing reaction between thewhole blood and antigen. Due to the allergy reaction basophils 13 in thewhole blood sample will be stimulated and will release histamine 8;

(4) The whole blood sample 11 having stimulated by the antigen 12 willbe sampled by means of a micro-syringe or a pipette.

(5) The sample will be added to the oocyte expressing histaminereceptors.

(6) The change in electric response of the oocyte will be measured bymeans of the apparatus shown in FIG. 1 to determine the change incurrent as have been described above.

(7) From the current change value, the concentration of histamine (A) inthe whole blood sample 11 having stimulated by the antigen 12 may bedetermined by referring to the predetermined correlation (calibrationcurve).

The releasing rate of histamine α (%) may be determined by

α=100×(A−C)/B

where B is a histamine concentration given by the quantitativemeasurement of histamine released from the cells in the sample wholeblood after freezing and thawing the sample having stimulated by theantigen, C is a concentration of free released histamine in nonstimulated state by adding the buffer solution 23 instead of the antigeninto the blood sample.

The concentration of free histamine released in non-stimulation Cdesignates to the concentration of free histamine released by thestimulation of buffer, i-e., so-called “concentration of correction ofbackground”.

The time required for a quantitative analysis of histamine in accordancewith the first preferred embodiment will be approximately 30 minutes,which is significantly faster when compared with 3 hours needed for themethod described in the Prior Art 3 above and 4 hours needed for themethod described in the Prior Art 4 above, resulting in a saving oftime.

The procedure of the method of measuring histamine in accordance withthe first preferred embodiment of the present invention is characterizedin that the collected sample may be used for quantitative analysiswithout need of any pretreatment of whole blood, with significantlyfewer steps of processing as compared with any of Prior Art. Theprocedure of the method in accordance with the present inventionrequires only histamine of known concentration at the time of making acalibration curve, and no other reagents are needed, allowing theanalytical cost to be reduced.

[second embodiment]

FIG. 5 shows an example of results of histamine analysis using wholeblood samples (5 samples), obtained by using the apparatus shown in FIG.1. The cedar pollen was used as antigen. FIG. 5 shows the results ofmeasurement of histamine, A (concentrations of histamine in the wholeblood samples stimulated by the cedar pollen), B (concentrations ofhistamine quantitatively determined from the histamine released from thecells of whole blood samples by freezing and thawing the whole bloodsamples stimulated by the cedar pollen) C (concentrations of freelyreleased histamine in non stimulated state derived from the cells in thewhole blood sample by adding the buffer solution 23 thereto instead ofthe cedar pollen antigen) and α (%) (histamine releasing rate), eachdetermined for respective sample in accordance with the proceduresimilar to that of the first embodiment as have been described above.

In the histamine-releasing test in general with respect to bloodsamples, when the histamine-releasing rate of a sample is higher than orequal to 10% when stimulating with a specific allergen, the sample maybe considered to be positive in the allergy reaction. In the exemplaryembodiment shown in FIG. 5, the samples 1 and 3 are to be consideredthat the allergy reaction for the cedar pollen has been occurred. Theseresults were consistent with the results of conventional HRT (HistamineRelease Test) method as well as the subjective symptom or examinees.

[third embodiment]

FIG. 6 shows a schematic diagram of an apparatus for measuring histaminein accordance with another preferred embodiment of the presentinvention, illustrating the overview of an apparatus for quantitativelyanalyzing histamine by using the oocyte with histamine receptorsexpressed and a whole blood sample.

The apparatus shown in FIG. 6 is consisted of, in addition to thecomponents of the apparatus shown in FIG. 1, a pouring tube 14 forpouring the buffer solution 23 into the vessel 1 with the assistance ofa pump and the like, a draining tube 15 for draining excessive buffersolution 23 from the vessel 1 with the assistance of a pump and thelike, and a draining tube 20 for purging the solution contained in thevessel 1 with the assistance of a pump and the like each time when thehistamine analysis of a sample is completed. It should be noted that inFIG. 6 the external electrode 5, the differential amplifier 6, thegrounding wire of differential amplifier 6, the recorder 7, the electricresponse of oocyte 10 are omitted for the sake of simplification.

The buffer solution 23 for oocyte 2 will be infused to the vessel 1through the pouring tube 14 in excess of the liquid level detectorportion of liquid level meter (not shown in FIG. 6) disposed inside thevessel. Then the driving apparatus of drain pump will be driven based onthe output from the level detector until the level of buffer 23 willreach to the level of the level detector sensing portion in order todrain the excessive buffer solution 23 from the vessel 1 through thedraining tube 15.

Thereafter, if for some reason or another the level of buffer solution23 contained in the vessel 1 is decreased or increased, the decrease ofbuffer solution 23 will be detected by the level detector sensor portionand the driving apparatus of drain pump will be driven in accordancewith the output of the level detector to automatically add an amount ofbuffer solution 23 through the pouring tube 14 into the vessel 1 inorder to maintain the level of buffer solution 23 at a predeterminedlevel. In this manner a predetermined constant amount of buffer solution23 sufficient for dipping the oocyte 2 will be held in the vessel 1.

As shown in FIG. 6, in the recess 16 at the bottom of the vessel 1filled with the buffer solution 23 for oocyte 2, a Xenopus oocyteexpressing histamine receptors 2 will be immersed in a static manner. Assimilar to the apparatus shown in FIG. 1, the fine tips of glasselectrode 3 and glass electrode 4 will be inserted into the Xenopusoocyte 2. The potential appeared in the glass electrode 3 will betransmitted to the differential amplifier 6 not shown in FIG. 6 as thedifferential potential from the external electrode 5. The differentialamplifier 6 receiving the potential signaled from the glass electrode 3will generate the current difference between the glass electrode 3signal and the fixed potential in the whole cell clamping method andapply it to the Xenopus oocyte 2 through the glass electrode 4. Themembrane potential of the oocyte will be accordingly held at apredetermined constant level of −60 mV.

Then the whole blood sample 11 prepared in accordance with the procedureshown in FIG. 4 will be added gently over the Xenopus oocyte 2 themembrane potential of which is held at −60 mV by means of an instrumentsuch as micro-syringe or pipette 9. The whole blood sample 11 added tothe vessel over the oocyte contains the histamine 8 released by thehistamine releasing reaction, antigens 12, basophils 13, plasma and thelike. The change of current before and after the histamine 8 is bound tothe histamine receptors will be measured. As have been described in theforegoing discussion, the concentration of histamine 8 released from thecells in the whole blood may be determined from the change of current bypredetermining a calibration curve.

Another configuration of apparatus for adding the prepared whole bloodsample 11 over the Xenopus oocyte 2 without using a micro-syringe orpipette 9 will be described below in greater details. As shown in FIG.6, a fine reacting tube (glass capillary) 17 may be provided to theapparatus for carrying out therein the histamine releasing reaction inthe whole blood sample, such that step (3) of the procedure shown inFIG. 4 will be completed within the fine reacting tube 17. On the innersurface of the fine reacting tube 17 the antigen 12 such as cedar pollenand the like are immobilized.

A temperature controlling device (heater) 18 configured so as tosurround the outer surface of tubing wall of the fine reacting tube 17will control the temperature of fine reacting tube 17 at a temperatureranging from 30° C. to 45° C. for promoting the histamine releasingreaction. 5 to 20 mL of whole blood sample will be directly poured intothe fine reacting tube 17 held at 37° C. via the tubing of left handside of a cock 22 in the figure and the sample will be held therein for30 minutes to initiate and promote the histamine releasing reaction.

Once the histamine releasing reaction in the fine reacting tube 17 hasbeen completed, the temperature inside the fine reacting tube 17 will bedecreased back to the room temperature and then a cock 19 will be openedso that the blood sample solution containing the histamine 8 released bythe histamine releasing reaction, antigen 12, basophils 13, plasma andthe like will be transferred to the surface of the Xenopus oocyte 2 heldat the recess 16 of the bottom of vessel 1 through the cock 19 andflowing tube 21. Through the fine tip of the flowing tube 21, thesolution containing histamine 8 and the like will be added over theXenopus oocyte 2. After transfer the cock 19 may be closed.

The cross-sectional form of the flowing tube 21 is round and the tube 21is bent at the proximity of its tip. The flowing tube 21 will beinserted to an opening provided on the sidewall of the vessel 1 in roundcross-sectional shape. The axis of the opening aligns to the projectionline extendingly projected to the bottom of vessel 1, the axis passingthrough the center of recess 16. The flowing tube 21 may be movable inrotative and translatory manner within the opening. The relativeposition of the tip of flowing tube 21 with the surface of oocyte heldon the recess 16 may be adjusted by moving the flowing tube 21 inrotative or translatory direction as described above.

After the adjustment of relative position, the sample solutioncontaining the histamine 8, antigen 12, basophils 13, plasma and thelike will be gently add the Xenopus oocyte 2 having the membranepotential held at −60 mV by controllably flowing the buffer solution 23or sterile gas from the tubing of left hand side of the cock 22 with theflow rate being controlled.

As have been described above, by measuring the change in current beforeand after the histamine 8 is bound to the histamine receptors of oocyteand using the predefined calibration curve, the concentration ofhistamine 8 released from the cells in the whole blood sample may bedetermined.

When the measurement of change in current has been completed, the glasselectrodes 3 and 4 may be drawn from the oocyte, and the solution in thevessel 1 together with the oocyte may be drained through the drainingtube 20 by for example suction by means of a purge pump. Then theelectrodes 3 and 4 may be moved in one side of recess on which theoocyte will be held. Some washing solution may be poured into the vessel1 through the pouring tube 14 to stir washing solution with a stirrernot shown in the figure in the vessel 1 to wash and rinse the vessel andthereafter the washing solution may be drained through the draining tube20. The above washing process will be repeated three to four times tocomplete washing step of the vessel.

Then, by opening the cock 19 to flow washing solution through the tubingof left hand side of the cock 22 for several times to wash and rinse theinside of the fine reacting tube 17, cock 19, and flowing tube 21.Finally, the above washing process will be iterated for several times tocomplete washing step of the apparatus. In the configuration using thefine reacting tube 17, the quantitative analysis of the concentration ofhistamine released from the cells in the whole blood sample along withthe histamine releasing reaction of whole blood may be continuouslycarried out. More specifically, the sequential change of histaminereleasing process may be allowed to measure in a contiguous manner. Insuch a case the relative position of the fine tip of flowing tube 21 andthe oocyte held in the recess 16 should be controlled by the rotativeand translative displacement as have been described above prior toanalysis.

Then the sample will be directly injected through the tubing of lefthand side of the cock 22 to the fine reacting tube 17 having the antigen12 such as cedar pollen immobilized on the surface of inner wall thereofand held by the temperature controlling device (heater) 18 at atemperature in a range from 30° C. to 45° C. for promoting the histaminereleasing reaction. Immediately after that, the cock 19 will be openedto flow the buffer solution 23 held at a temperature approximately sameto that of the fine reacting tube 17 at slower rate through the tubingof left hand side of the cock 22.

A membrane having a number of micropores through which the histamine 8may be passed but the basophils 13 may not be provided for examplebetween the fine reacting tube 17 and the cock 19.

Although the present invention has been described in conjunction withseveral preferred embodiments thereof, it should be understood thatthese embodiments are disclosed by way of examples and the presentinvention is not to be limited thereto. It should be recognized thatmany changes and modifications may be made by those skilled in the artwithout departing from the true spirit and the scope of the presentinvention according to the appended claims.

What is claimed is:
 1. A histamine measuring method for quantitativelyanalyzing a concentration of histamine, comprising the steps of:preparing an oocyte which expresses histamine receptors and identifiesspecifically the presence of histamine; holding said oocyte in a recessat a bottom of a vessel in which a buffer solution is filled; insertingfirst and second electrodes into said oocyte held in the recess;measuring a potential of a membrane of said oocyte by means of saidfirst electrode inserted into said oocyte held in the recess, andmaintaining the potential of the membrane of said oocyte held in therecess at a constant potential by applying a current through said secondelectrode inserted into said oocyte held in the recess; infusing asample into a fine reacting tube having an antigen immobilized on aninner surface thereof wherein said antigen stimulates histamine releasefrom the sample infused into said fine reacting tube; transferring asolution containing histamine released from the sample in said finereacting tube to said vessel to make contact with said oocyte in saidvessel; detecting a change in an electric response of said oocyte beforeand after binding of histamine to the histamine receptors; anddetermining a concentration of histamine released from the sample insaid fine reacting tube.
 2. A histamine measuring method according toclaim 1, wherein said sample is a whole blood sample withoutpretreatment or a mast cell suspension without pretreatment.
 3. Ahistamine measuring method according to claim 1, further comprising thestep of: controlling a temperature of said fine reacting tube at atemperature ranging from 30° C. to 45° C. to promote release ofhistamine from the sample.
 4. A histamine measuring method according toclaim 1, further comprising the step of: determining the concentration(A) of histamine released from the sample in said fine reacting tube bymeans of a predefined calibration curve obtained by stimulating saidoocyte with a plurality of known concentrations of histamine to detectthe electric response of said oocyte to obtain a correlation betweensaid electric responses and said plurality of known concentrations ofhistamine.
 5. A histamine measuring method according to claim 4, furthercomprising the step of: determining a histamine releasing rate given by(A−C)/B where B is a histamine concentration given by a quantitativemeasurement of histamine contained in cells in said sample and releasedby freezing and thawing the sample stimulated by the antigen, C is aconcentration of free histamine released from the sample withoutstimulation after adding some buffer solution instead of the antigeninto the sample.
 6. A histamine measuring method according to claim 1,wherein said oocyte is an unfertilized oocyte of Xenopus lavis.
 7. Ahistamine measuring method for quantitatively analyzing a concentrationof histamine, comprising the steps of: preparing a cell which expresseshistamine receptors and identifies specifically the presence ofhistamine; holding said cell in a recess at a bottom of a vessel inwhich a buffer solution is filled; inserting first and second electrodesinto said cell held in the recess; measuring a potential of a membraneof said cell by means of said first electrode inserted into said cellheld in the recess, and maintaining the potential of the membrane ofsaid cell held in the recess at a constant potential by applying acurrent through said second electrode inserted into said cell held inthe recess; infusing a sample into a fine reacting tube having anantigen immobilized on an inner surface thereof wherein said antigenstimulates histamine release from the sample infused into said finereacting tube; transferring a solution containing histamine releasedfrom the sample in said fine reacting tube to said vessel to makecontact with said cell in said vessel; detecting a change in an electricresponse of said cell before and after binding of histamine to thehistamine receptors; and determining a concentration of histaminereleased from the sample in said fine reacting tube.
 8. A histaminemeasuring method according to claim 7, wherein said cell is an oocyteand said antigen is a cedar pollen.
 9. A histamine measuring methodaccording to claim 8, wherein said oocyte is an unfertilized oocyte ofXenopus lavis.
 10. A histamine measuring method according to claim 7,wherein the sample is whole blood, leukocytes in blood, or a mast cellsuspension.
 11. A histamine measuring method according to claim 7,further comprising the step of: controlling a temperature of said finereacting tube at a temperature ranging from 30° C. to 45° C. to promoterelease of histamine from the sample.